Hyperglycemia and increased transforming growth factor-? (TGF?) expression activate mTOR (mechanistic target of rapamycin), which acts as a key node to promote glomerular and tubular hypertrophy and matrix protein fibronectin expression. We have reported that high glucose and TGF? activate both mTOR complexes (C1 and C2) in mesangial and proximal tubular epithelial (PTE) cells and in kidneys of type 2 and type 1 diabetic mice. Blocking mTOR activity with rapamycin, we showed reduction in albuminuria, renal hypertrophy and matrix expansion in these models of diabetes. Complete inhibition of mTOR by rapamycin can be detrimental as the activity of this kinase is required for maintaining renal cell homeostasis. Also, rapamycin changes gene expression profile, which causes adverse effects. Therefore, alternative therapeutic approach is necessary to block mTOR kinase. Our data demonstrate that the expression of deptor, a component of both mTOR complexes, and inhibitor of their kinase activities, is reduced in kidneys of diabetic rodents and in mesangial and PTE cells treated with high glucose or TGF?. Exploiting how deptor is suppressed to increase mTOR activity can identify novel molecular targets to block diabetic renal complications. In our preliminary data, we find reduction in deptor levels concomitant with increased expression of EZH2 (enhancer of zeste homolg 2), the catalytic subunit of the polycomb repressor complex 2, which trimethylates histone H3 at lysine- 27 to block transcription of a specific gene. Moreover, we show enhanced expression of the E3 ubiquitin ligase ?TrCP (transducin repeat containing protein) by high glucose and TGF? in renal cells. ?TrCP targets deptor for degradation. Furthermore, we demonstrate high glucose and TGF? increase the expression of a family of microRNA, miR-181 (a,b,c,d), and miR-221. These microRNAs are also increased in kidneys of type 1 and type 2 diabetic mice and target deptor for downregulation. In this proposal, using cultured mesangial and PTE cells and renal tissues from diabetic OVE26 and db/db mice, we will test the hypothesis that inappropriate deptor downregulation contributes to renal hypertrophy and matrix expansion in diabetic kidney disease. Probing the specificities of underlying mechanisms, we will define the regulatory modules of deptor suppression. In the first specific aim, we will investigate EZH2 as a candidate for transcriptional suppression of deptor, hypertrophy and, fibronectin and PAI-1 (plasminogen activator inhibitor-1) expression. In the second aim, the role of ?TrCP in deptor protein degradation/mTORC1/C2 activation and its cross talk with TGF? signaling in forcing mesangial and PTE cell hypertrophy and, fibronectin and PAI-1 expression will be examined.
In specific aim 3, we will investigate the contribution of miR-181 family and miR-221 to hypertrophy and, fibronectin and PAI-1 expression in response to high glucose and TGF? in mesangial and PTE cells and in diabetic mice kidneys. To address these aims, techniques including immunoblotting, immunoprecipitation, qRT-PCR, morphometry, immunohistochemistry, reporter transfection assays, chromatin immunoprecipitation assays and siRNA-mediated downregulation of proteins will be used.
A recent study showed that 29% of the hospitalized veterans had chronic kidney disease, which often leads to end stage renal disease (ESRD). Diabetes is one of the most prevalent chronic diseases among people aged 20 years and older in the US. The demographics of veteran population fall in this age group. 23.1% of people in the age group of 60 years or older have diabetes. Diabetic kidney disease is the leading cause of ESRD, accounting for about 44% of new cases each year. Current therapies only delay the progression to ESRD and do not arrest the disease.Thus there is an urgent need for therapeutic approaches, which may include promoting protective or regenerative factors and blocking mechanisms of injury. Renal cell hypertrophy and matrix expansion are two pathological features of diabetic kidney disease. This proposal will explore novel signaling protein, deptor, which can be targeted using small moleucule inhibitors and anti- microRNAs in the affected patients, who suffer from kidney disease resulting from complications of diabetes.
Showing the most recent 10 out of 12 publications
